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. 2016 May 1;129(9):1792-801.
doi: 10.1242/jcs.180737. Epub 2016 Mar 17.

Ca2+ Is a Key Factor in α-Synuclein-Induced Neurotoxicity

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Free PMC article

Ca2+ Is a Key Factor in α-Synuclein-Induced Neurotoxicity

Plamena R Angelova et al. J Cell Sci. .
Free PMC article

Abstract

Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca(2+) signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca(2+) in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca(2+) signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca(2+), which prevented the α-synuclein-induced Ca(2+) dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca(2+) signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca(2+) dysregulation and Ca(2+)-dependent cell death.

Keywords: Ca2+ signalling; Neuronal death; Parkinson's disease; α-Synuclein.

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Application of extracellular α-synuclein induces a cytosolic Ca2+ signal. (A) A characteristic single-molecule FRET histogram of the 29-h time-point oligomerisation was used in this study. There are both globular non-toxic (18%, E=0.41, blue line), and toxic β-sheet-containing oligomers (82%, E=0.62), red and black lines, present. (B) Application of 40 nM oligomeric α-synuclein (α-syn) induces a Ca2+ signal in an acute rat brain slice. (C,D) 40 nM of monomeric (C) or oligomeric (D) α-synuclein induced an elevation of cytosolic Ca2+ signal in primary neurons and astrocytes. Both forms of α-synuclein induced a rise in basal cytosolic Ca2+ as well as Ca2+ spikes. (E) Dose–response experiment demonstrating the number of cells responding at different concentrations of oligomeric α-synuclein. (F) Histogram demonstrating the proportion of cells exhibiting each pattern of Ca2+ signal. (G) The α-synuclein-induced Ca2+ signal was also observed in neurons derived from iPSCs. (H) Enriched oligomeric α-synuclein induced a rise in basal cytosolic Ca2+ as well as Ca2+ spikes. Results are mean±s.e.m. [n=98 for monomers; n=183 for oligomers (E,F)].
Fig. 2.
Fig. 2.
Identification of the source of the α-synuclein-induced Ca2+ signal. (A,B) Both monomeric and oligomeric Ca2+ signals could be prevented completely by removal of Ca2+ from the extracellular medium. (C) Inhibition of PLC by U73122 and inhibition of IP3 receptors by Xestospongin C had no effect on the α-synuclein-induced Ca2+ signal. (D,E) Depletion of the ER Ca2+ store using thapsigargin did not affect the monomeric or oligomeric-induced Ca2+ signal. (F) Representative image of single neuron following application of equimolar ratio of α-synuclein labelled with monomeric AF488 (green) and AF647 (red) demonstrating intracellular uptake of recombinant α-synuclein species. Scale bar: 10 μm.
Fig. 3.
Fig. 3.
Mn2+ quench assay confirms that α-synuclein-induced Ca2+ transients originate from a Ca2+ influx across plasmalemmal membrane. (A,B) Fura-2-loaded neurons showed typical [Ca2+]c fluctuations in response to monomeric (A) or oligomeric (B) α-synuclein. With the addition of 40 µM Mn2+, each [Ca2+]c transient was accompanied by a step quench of the 360-nm fura-2 signal, confirming that each transient reflects a pulsed influx of divalent cations seen in response to α-synuclein. (C) Application of monomeric α-synuclein in the absence of MnCl2 did not induce any alteration in the fura-2 360-nm signal. (D) The α-synuclein-induced Ca2+ influx was independent of the presence of inhibitors of plasmalemmal channels [20 µM verapamil, 1 µM Nifedipine, 10 µM MK-801, 20 µM CNQX or 50 µM (S)-MCPG].
Fig. 4.
Fig. 4.
Monomeric α-synuclein leads to impairment of Ca2+ efflux. Application of 5 μM glutamate to cells pre-incubated with α-synuclein monomers (A) resulted in significantly delayed recovery of the cytosolic Ca2+ signal (over a period of 10 min) compared to control (recovery within 2 min, B). Results are mean±s.e.m. [n=91, control; n=68, monomers (B)]
Fig. 5.
Fig. 5.
Channel activity of α-synuclein on artificial membranes. (A) Oligomeric α-synuclein was added to the cis compartment of a bilayer cuvette to a final concentration of 100 nM. The membrane was suspended between symmetric HBSS supplemented with 1 mM CaCl2. (B) Channels induced by 100 nM oligomer α-synuclein in lipid bilayers suspended between aqueous solution of 15 mM NaCl (cis) and 150 mM NaCl (trans), 2 mM CaCl2, 10 mM Tris-HCl pH 7.4 (symmetric). Voltage of 150 mV was applied across BLM. (C) Current–voltage dependence of the channels presented in B. (D) Irregular currents induced by 100 nM of monomeric α-synuclein in BLMs. The membrane was suspended between symmetric HBSS supplemented with 1 mM CaCl2.
Fig. 6.
Fig. 6.
Protective effect of Ca2+-free medium against α-synuclein-induced caspase-3 activation and cell death. (A,B,D) 40 nM oligomeric α-synuclein significantly activates the NucView 488 caspase 3 substrate in neurons and astrocytes. (C) Pre-incubation in Ca2+-free medium significantly reduced caspase 3 activation, shown as the time from addition of the peptide to activation of the substrate (i.e. increase of fluorescence in A). (D) Quantification of α-synuclein-induced apoptosis in Ca2+-containing and Ca2+-free medium. (E) Cell death was assessed using propidium iodide to label dead cells and Hoechst 33342 to label all cells. (F) Quantification of α-synuclein-induced cell death in normal and Ca2+-free medium. Results are mean±s.e.m. [n=69 (D); n=32 (norm); n=37 (Ca2+-free) (F)]. ***P<0.001; ns, not significant (Student's t-test).

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